FN Archimer Export Format
PT J
TI Microbial enzymatic assays in environmental water samples: impact of Inner Filter Effect and substrate concentrations
OT Méthodologie des activités enzymatiques microbiennes en milieu aquatique : impact du filtre interne et des concentrations en substrat
BT 
AF URVOY, Marion
   LABRY, Claire
   DELMAS, Daniel
   Creac'h, Layla
   L HELGUEN, Stephane
AS 1:1,2;2:1;3:1;4:1;5:2;
FF 1:;2:PDG-ODE-DYNECO-PELAGOS;3:PDG-ODE-DYNECO-PELAGOS;4:;5:;
C1 Ifremer, DYNECO, F-29280 Plouzané, France
   Univ Brest, CNRS, IRD, Ifremer, LEMAR, F-29280 Plouzané, France
C2 IFREMER, FRANCE
   UBO, FRANCE
SI BREST
SE PDG-ODE-DYNECO-PELAGOS
UM LEMAR
IN WOS Ifremer UPR
   WOS Cotutelle UMR
   copubli-france
   copubli-univ-france
IF 2.634
TC 7
UR https://archimer.ifremer.fr/doc/00653/76524/77614.pdf
LA English
DT Article
AB As microbial enzymatic activities initiate the mineralization of organic matter through the microbial loop, it is important to correctly measure those activities and be able to perform inter-study comparisons. Enzymatic activity assays are typically carried out using fluorogenic substrate analogs, such as 4-Methylumbelliferone and 7-Amino-4-methylcoumarin linked to sugar monomers, phosphate group or amino acids. However, methodological divergences can be found in aquatic science literature, potentially leading to misestimated activities. To highlight some of those methodological key points, we first addressed the potential occurrence of an Inner Filter Effect (IFE), a fluorometric artifact that affects the relationship between fluorophore concentration and fluorescence intensity, due to absorption of exciting or emitted light. It has never been considered in the context of environmental waterstudies before, despite significantly affecting measured activities. IFE occurred with 2 out of 3 tested spectrofluorometers when assaying proteases, although no IFE was detected for phosphatase assays. We also evaluated how substrate concentration ranges might affect kinetic parameters estimation, revealing that a many existing studies might use insufficient maximum substrate concentration. Finally, for single substrate concentration assays, we argued for the use of saturating substrate concentration, as naturally occurring substrates might compete with the fluorogenic analog at trace level. The amendment of a molecule mimicking natural substrates generated a significant inhibition of natural seawater phosphatases and proteases assayed with trace concentrations of fluorogenic substrate, while almost no inhibition occurred at higher concentrations. Those key points need to be addressed in order to assess enzymatic rates and allow inter-study comparison.  
PY 2020
PD DEC
SO Limnology And Oceanography-methods
SN 1541-5856
PU Association for the Sciences of Limnology and Oceanography (ASLO)
VL 18
IS 12
UT 000579826900001
BP 725
EP 738
ID 76524
ER

EF